Underlying principles

■ Use of animals can benefit society

■ But it should be done so as to minimize distress

■ Mostly an ethical issue

■ But also, a distressed animal produces unreliable experimental results

■ The decision to approve a proposed use of animal subjects involves weighing:

■ The amount of unavoidable distress

■ The potential societal benefit

Justification for using animals

■ I. Why is this the right animal model?

■ A. Research should be undertaken with a clear scientific purpose.

■ B. There should be a reasonable expectation that the research will:

• 1. Increase knowledge of the processes underlying the evolution, development, maintenance,

alteration, control, or biological significance of behavior

• 2. Increase understanding of the species under study

• 3. Provide results that benefit the health or welfare of humans or other animals.

■ II. Does an animal need to be used?

■ i.e., are there alternative models?

■ III. What is the minimal number of animals needed?

Transportation Issues

■ I. Proper packaging

■ II. Timely shipping

■ III. Appropriate care upon arrival

■ Once the animals arrive:

■ Adequate Veterinary Care

■ Consultation with the principal investigator to minimize pain and distress.

What is “distress”?

■ Pain, suffering, fear

■ Assumptions that:

■ Invertebrates experience distress less than vertebrates.

■ “Lower” vertebrates experience distress less than more advanced ones, because simpler brains have
less capacity to remember past events and to conceptualize and anticipate future events.

PI Responsibilities

■ A. Minimize pain and distress

■ 1. Appropriate pain relieving drugs

• Unless withholding such drugs is justified for scientific reasons, in writing, by the principal investigator
and will continue only for the necessary period of time.

■ 2. Not include the use of paralytics without anesthesia.

(This is a flat prohibition).

■ 3. Animals that would otherwise experience severe or chronic pain or distress that cannot be relieved
will be painlessly euthanized.

■ 4. The animals living conditions will be appropriate for their species in accordance with standards that
will contribute to their health and comfort.

■ 5. Medical care for animals will be available and provided as necessary by a qualified veterinarian.

■ 6. Activities that involve surgery must include appropriate provision for pre-operative and post-
operative care of the animals in accordance with established veterinary medical and nursing practices

7. All survival surgery will be performed using aseptic procedures, including surgical gloves, masks,
sterilized instruments, and aseptic techniques.

■ 8. No animal will be used in more that one major operative procedure from which it is allowed to
recover unless:
• a. Justified for scientific reasons by the principal investigator, in writing.

• b. Required as routine veterinary procedure or to protect the health or well-being of the animal as
determined by the attending veterinarian.

■ B. Ensure that personnel conducting procedures on the species being maintained or studied are
appropriately qualified and trained in those procedures.

■ C. Humane methods of animal maintenance and experimentation.

■ 1. The basic needs of each species of animal.

■ 2. Proper handling and care for the various species of animals used by the facility

■ 3. Proper pre-procedural and post-procedural care of animals.

■ 4. Aseptic surgical methods and procedures.

C.

■ 1. The concept, availability, and use of research or testing methods that limit the use of animals or
minimize animal distress

■ 2. Proper use of pain relieving drugs for any species of animals used by the facility.

■ 3. Methods whereby deficiencies in animal care and treatment are reported, including deficiencies
reported by facility employees

Housing Standards

■ I. Heating, cooling, and temperature

■ II. Ventilation

■ III. Compatible groupings

■ IV. Exercise

■ V. Food and water

■ VI. Psychological well-being

Disposal

■ The return of wild-caught animals to the field can carry substantial risks

■ To the formerly captive animals and to the ecosystem.

■ Animals reared in the laboratory should not be released

• In most cases, they cannot survive or they may survive by disrupting the natural ecology.

■ Proper (humane) euthanasia

High-quality cages

INDIVIDUALLY VENTILATED CAGES

laminar air

Diets

!Chemically defined diets

!Custom-mix diets

!Natural diets

!Purified diets

!Semi-purified diets

!Other diets

Bedding

! Contact bedding solid-bottom cages

!Noncontact bedding suspended-wire cages

!Ground corncobs

!Sawdust
!Pelleted peanut hulls

Bedding materials

• ! Pelleted alfalfa

• !Processed wood chips

• !Processed paper chips

Water

deionization osmosis

!Fresh uncontaminated and sterile water

!Water purification systems reverse ultrafiltration

!Hyperchlorination/acidification

Table 1.0.2 Recommended Relative Humidity and Dry Bulb Temperature for Animals Housed inCagesa

!Regular diurnal light cycle

!Light levels: 323

lux (30-foot candles)

! 1 meter above the floor

Mouse Handling and Manual Restraint

Mouse handling and manual restraint. Apply slight, rearward traction on the tail

(A). Grasp skin behind ears with thumb and index finger (B). Transfer the tail from the preferred hand to
beneath the little finger of the hand holding the scruff of the neck (C).
Reproductive Data Of Mice

• Stage

• Sexual maturity

• Estrous cycle

• Gestation

• Weaning age

• Reproductive life

• Life span

• Pups begin eating dry food

Minimizing Pain and Distress

Causes of Pain and Distress in Mice

• Research staff should be familiar with the causes of animal pain and distress. Pain and distress are
caused by spontaneous and experimentally-induced disease or injury.

• Many factors may contribute to an animal’s distress or discomfort, including extreme homeostatic
challenges.

Factors in Pain & Distress

Changes in the following parameters may cause or be associated with animal

pain or distress:

! temperature (environmental and body temperature)

! hypoxia

! edema

! blood electrolytes, e.g. hyperkalemia

! dehydration

! environment
! caging

! cage mates

! lighting

! humidity

! noise

! Vibration

Note - Smaller mammals experience physiologic changes such as starvation (due to high metabolic rate)
and chilling (due to large ratio of body surface area to mass) faster than larger animals.

Signs of Pain and Distress

• Signs of pain and distress in rodents are not easy to detect because of their body size, their tendency
to conceal outward signs of pain and distress, and their habit of hiding or freezing when disturbed.

• Nevertheless, signs of pain or distress can be detected in rodents by carefully observing subtle changes
in behavior.

• The ability to properly assess pain and distress in rodents requires:

--knowledge of normal rodent behavior and appearance

--a systematic approach to observing clinical signs in rodents

Assessing Appearance and Behavior

From the Cage Exterior

• Routinely inspect the rodents through the top and sides of the cage. Get in the habit of removing the
cage from the shelf and looking through all sides of the cage. Signs of distress may be missed in animals
on lower or upper shelves because of low lighting or difficult access.

• Baby mice and rats can be inconspicuous within piles of bedding or nestboxes.

Cage Wirelid Off

• Lift the cage wirelid to elicit a response to your presence. This disturbance may prompt the animals to
move about the cage. Examine the animals’ behavior, gait, and hair coat.
• Normal mice are inquisitive and explore their cage perimeter.

Behaviors to Observe

• Abnormal mice may huddle in their cage, or they may fail to move around and explore their cage. In
addition, mice may vocalize when approached.

• Inspect the animal’s mode and speed of movement.

Observe the tail position when the animal moves.

• Is the gait (how it walks) awkward? Observe how all limbs move while walking.

• Does the animal teeter or stumble?

• Is the animal’s back hunched and abdomen tucked while walking?

• Is the tail held stiff and upright? Or does the tail drag?

Tip: Observe a cage of normal animals for a comparison.

• Examine (and treat) an individual mouse by gently restraining the animal. You can move the animal to
a separate examination box for detailed clinical inspection.

Types of Observations

A common approach to assessing animal appearance and behavior is through observation of the
following:

! Activity Level

! e.g., hypoactivity (hunched, huddled, lethargic), hyperactivity, restlessness, lack of inquisitiveness

! Attitude

! e.g., arousal, depression, awareness of surroundings

! Behavior, Spontaneous

! e.g., vocalization, self-trauma, isolation from cage mates. These observations are made without
disturbing the animal.

! Behavior, Provoked
! e.g., vocalization, hiding, aggressiveness, minimal response. These observations are made when the
animal is disturbed or even prodded.

Types of Observations

! Body Condition

! e.g., emaciation, missing anatomy

! Food and Fluid Intake

! e.g., elimination of feces and urine

! Fur and Skin

! e.g., unkempt or greasy or dull fur; porphyrin staining around eyes and nostrils;

cyanotic, pale, or congested mucous membranes or skin (ears, feet, tail); skin lesions; soiled anogenital
area

! Eyes

! e.g., clarity/condition of lens, cornea; position of globe (e.g., sunken in orbit or protruding); condition
of eyelids, encrustation

! Posture

! e.g., hunched back, tucked abdomen; prostrate; head tucked

Physical Examination

You may evaluate:

• Behavior

• Body weight

• Surface lesions (wounds, masses)

• Hydration status

• Body temperature (telemetric methods)

• Blood parameters (Blood collection can be difficult/ stressful in mice; may be used to confirm disease
or failed treatment.)
• For specific recommendations on evaluating these parameters and supportive procedures, refer to the
AALAS Learning Library course Post-Procedure Care of Mice and Rats in Research: Minimizing Pain and
Distress.

Euthanasia

! CO2 60-100% (30 sec) neonates resistance

! Cervical dislocation (Expert)

! Chloroform

Zebrafish (Danio rerio)

! Popular model in biomedical research and emerging as a new important species for studying
mechanisms of brain function and dysfunction (Stewart, et al., 2014).

! Cold Spring Harbour Conference (1994) –

What is a zebrafish?

! Small freshwater fish from South Asia.

! 4 cm long when fully grown.

! Dark - light cycle : 10h : 14h

! Very easy to rearing.

! powerful experimental transgenic approaches based on microinjection of antisense morpholino

oligonucleotides (MOs) Image: Wikimedia

Why use zebrafish?

!Small size.

! All major organs present within 5 days post fertilisation.

!Short generation time (3-4 months).

Orthologue genes shared (Kerstin Howe et al)

Valid Criteria (Lammer, et al., 2009)

(1) The fertility rate of the parent generation should be ≥70%.

(2)The dissolved oxygen concentration should be ≥60% of the air saturation value at the beginning of
the test.

(3)The water temperature should be maintained at 28±1 °C in test chambers at any time during the test.

(4)Overall survival of embryos in the negative control and, where relevant, in the solvent

Larva handling

! E3 medium (for zebrafish embryos) : 34.8 g NaCl1.6 g KCl5.8 g CaCl2·2H2O9.78 g MgCl2·6H2OTo

prepare a 60X stock,

• dissolve the ingredients in H2O, to a final volume of 2 L. Adjust the pH to 7.2 with NaOH. Autoclave. To
prepare 1X

• medium, dilute 16.5 mL of the 60X stock to 1 L. Add 100 μL of 1% methylene blue (Sigma-Aldrich).

! Static flow

! Change medium daily

Adult Zf Maintenance

1. Fresh water

2. Temp 25 – 31 °C

3. Feeding 2-3 times/day

4. Food : Artemia (embryo), pellet/ flakes (adult)

5. photoPeriod : 14 light, 10 dark

Euthanasia

! Deepfreeze

! Cold water

! Tricane